Published on Web 08/19/2008
Hydroxylase Activity of Met471Cys Tyramine β-Monooxygenase Corinna R. Hess,†,‡ Zinian Wu,†,‡ Adora Ng,†,‡ Erin E. Gray,§ Michele A. McGuirl,§ and Judith P. Klinman*,†,‡ Departments of Chemistry and Molecular and Cell Biology, UniVersity of California, Berkeley, California 94720-3220 and Department of Chemistry, UniVersity of Montana, Missoula, Montana 59812 Received February 7, 2008; E-mail:
[email protected] Abstract: A series of mutations was targeted at the methionine residue, Met471, coordinating the CuM site of tyramine β-monooxygenase (TβM). The methionine ligand at CuM is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TβM mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type TβM in the oxidized enzyme forms. EPR spectroscopy suggests that the CuII coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys TβM. Met471Cys TβM provides the first example of an active mutant directed at the CuM site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.
1. Introduction
The neuroregulatory enzymes peptidylglycine R-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM) are among the few examples of copper-containing oxygenases. These enzymes employ two noncoupled mononuclear Cu centers, CuM and CuH, to carry out the hydroxylation of their substrates: peptidylglycines, dopamine, and tyramine for PHM, DβM, and TβM, respectively.1,2 Dioxygen activation, the first step in this mechanism, occurs solely at the CuM site; the CuH site functions as an electron transfer site, supplying the additional electron required for the oxidation of substrate. Dioxygen binds and reacts with the reduced CuIM site to generate what is commonly believed to be a CuIIM-superoxide species, which subsequently abstracts a hydrogen atom from the bound substrate.3,4 The question of how a mononuclear Cu center can effectively catalyze both O2 and C-H activation has sparked the interests of synthetic chemists as well.4,5 Several mononuclear copper complexes are known to react with dioxygen to form 1:1 Cu/ O2 adducts, analogous to the reaction at CuM.6 However, these complexes tend to be unreactive toward hydroxylation reactions. †
Department of Chemistry, University of California. Department of Molecular and Cell Biology, University of California. § University of Montana. (1) Klinman, J. P. J. Biol. Chem. 2006, 281, 3013–3016. (2) Gray, E. E.; Small, S. N.; McGuirl, M. A. Prot. Exp. Purif. 2006, 47, 162–170. (3) Evans, J. P.; Ahn, K.; Klinman, J. P. J. Biol. Chem. 2003, 278, 49691– 49698. (4) Chen, P.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 4991–5000. (5) Tolman, W. B. J. Biol. Inorg. Chem. 2006, 11, 261–271. (6) Cramer, C. J.; Tolman, W. B. Acc. Chem. Res. 2007, 40, 601–608. ‡
10.1021/ja800408h CCC: $40.75 2008 American Chemical Society
The unique coordination environment at the CuM site presumably accounts for much of the enzymes’ reactivity toward O2. Two histidines, one to two H2O/OH molecules, and a weakly bound methionine residue (Met471 in TβM) ligate the tetragonal CuM center.7,8 The methionine sulfur forms a 2.24 Å bond with the reduced Cu site but is absent from the metal coordination sphere in the oxidized enzyme form.9 Several studies have suggested the importance of this methionine ligand in regulating O2 binding and the following CuI-O2 a CuII-O2- equilibrium;3,4 the effect of a sulfur group on O2 binding and dissociation from the metal center also has been illustrated with Cu-β-diketiminate complexes.10 Furthermore, XAS studies and DFT calculations on PHM have implied that the dynamics of the CuM-S bond are critical to the stabilization of postulated CuM-O2 intermediates in the enzymes’ catalytic cycle.4,11 Studies to directly assess the function of the thioether ligand in the enzymes have been limited, however. The only isolated and well-characterized hydroxylase in which the relevant CuMmethionine ligand has been altered is the Met314Ile mutant of PHM.12,13 Replacement of Met314 by an isoleucine residue in (7) Blackburn, N. J.; Hasnain, S. S.; Pettingill, T. M.; Strange, R. W. J. Biol. Chem. 1991, 266, 23120–23127. (8) Prigge, S. T.; Kolhekar, A. S.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science 1997, 278, 1300–1305. (9) Blackburn, N. J.; Rhames, F. C.; Ralle, M.; Jaron, S. J. Biol. Inorg. Chem. 2000, 5, 341–353. (10) Aboelalla, N. W.; Gherman, B. F.; Hill, L. M. R.; York, J. T.; Holm, N.; Young, V. G.; Cramer, C. J.; Tolman, W. B. J. Am. Chem. Soc. 2006, 128, 3445–3458. (11) Bauman, A. T.; Jaron, S.; Yukl, E. T.; Burchfiel, J. R.; Blackburn, N. J. Biochemistry 2006, 45, 11140–11150. (12) Eipper, B. A.; Quon, A. S. W.; Mains, R. E.; Boswell, J. S.; Blackburn, N. J. Biochemistry 1995, 34, 2857–2865. J. AM. CHEM. SOC. 2008, 130, 11939–11944
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Met314Ile PHM resulted in the complete loss of enzyme activity. Significant changes in protein stability and the CuH coordination geometry were an additional consequence, further suggesting a structural role for the thioether ligand in the enzyme.14 We now have generated a series of mutations directed at the corresponding CuM-thioether ligand in TβM, Met471, to probe the importance of the methionine residue for catalysis by the Cu hydroxylases and to determine whether other residues at this site might support activity. TβM shares significant homology with its mammalian counterparts, DβM and PHM, and kinetic studies have demonstrated the mechanisms of the three enzymes to be very similar.1,15 Given that a high level expression system is unavailable for DβM and that PHM is expressed in mammalian cell lines, the rapid, high-yielding expression of TβM in Drosophila S2 cells is ideally suited for mutagenesis studies. Three mutations of the CuM-thioether ligand in TβM were investigated: Met471His, Met471Asp, and Met471Cys. The alternate residues at Met471 were expected to modify the redox and/or coordination environment of the CuM site and, consequently, affect copper binding and/or dioxygen and substrate activation by the enzyme. DFT calculations for a series of inorganic model complexes have demonstrated the varying effects of anionic and neutral donor groups on dioxygen coordination and activation at mononuclear Cu centers;16 the alternate residues were chosen with the aim of studying similar effects in the enzyme. In the present work, the copper binding stoichiometries, EPR spectroscopy, and activities of the TβM mutants were investigated and compared to the wild type enzyme. All three mutants were found to maintain the ability to bind Cu at both sites, and the EPR spectra point to similar coordination geometries for the oxidized enzymes as for the wild type enzyme. However, only the Cys mutant is capable of substrate hydroxylation. The altered kinetics of Met471Cys TβM additionally signify inactivation of the enzyme during the reaction with substrate, further highlighting the singular role of the methionine ligand for enzymatic catalysis. These findings provide the first example of a functional ligand replacement at the conserved methionine in the PHM, DβM, TβM family of copper proteins. The demonstrated importance of a sulfur-containing ligand in catalysis is expected to impact theoretical and biomimetic investigations of dioxygen/C-H activation at mononuclear copper centers. 2. Experimental Section Materials. Drosophila Schneider S2 cells, insect cell growth media, and Drosophila Expression System were purchased from Invitrogen. Blasticidin was purchased from Sigma. Primers were custom ordered, HPLC-purified, from Operon. Chromatography media was purchased from GE Healthcare, except Talon affinity resin, which was purchased from BD Biosciences. Assay reagents (13) While CHO cell lines containing Met314Cys and Met314His PHM mutations also have been reported (Kolhekar, A. S.; Keutmann, H. T.; Mains, R. E.; Quon, A. S. W.; Eipper, B. A. Biochemistry 1997, 36, 10901–10909. ), neither of the mutant proteins was isolated and structurally characterized, and activity assays were carried out with spent media rather than using purified protein. (14) Siebert, X.; Eipper, B. A.; Mains, R. E.; Prigge, S. T.; Blackburn, N. J.; Amzel, L. M. Biophys. J. 2005, 89, 3312–3319. (15) Hess, C. R.; McGuirl, M. A.; Klinman, J. P. J. Biol. Chem. 2008, 283, 3042–3049. (16) Gherman, B. F.; Heppner, D. E.; Tolman, W. B.; Cramer, C. J. J. Biol. Inorg. Chem. 2006, 11, 197–205. 11940
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were purchased from Sigma-Aldrich, except catalase, which was obtained from Roche. Protein Expression and Purification. Wild type and TβM mutants were expressed as the His-tagged constructs in Drosophila S2 cells as described previously.2 Met471 mutations were generated by PCR using the pBipTBM plasmid and primers encoding ∼20 bases upstream and downstream of the mutation. The forward primers for the mutants are shown below, with the mutated codon in bold; reverse primers were complementary. Met471Asp: 5′-GATTCTCCATCAGCGATGAGGATTGCGTCAACTATATCCAC Met471Cys: 5′-GATTCTCCATCAGCGATGAGTGCTGCGTCAACTATATCCAC Met471His: 5′-GATTCTCCATCAGCGATGAGCACTGCGTCAACTATATCCACTAC The resulting altered pBipTBM plasmid was transformed into Escherichia coli strain NovaBlue (Novagen) or XL1 Blue (Stratagene) cells and purified using the Qiagen Highspeed Midiprep Kit. The composition of the purified plasmid was confirmed by automated DNA sequencing (University of California, Berkeley, Sequencing Facility and Murdock DNA Sequencing Facility, University of Montana), prior to transfection into Schneider S2 cells. Cells were harvested, and the protein was purified using anion exchange, His-tag affinity, and size exclusion chromatography as described previously for WT TβM. High purity fractions (single banded, as determined using SDS-PAGE, and comparison to WT enzyme in the case of the mutants) were pooled, and protein concentrations were determined by UV absorbance at 280 nm. TβM molecular weights and extinctions coefficients were determined using ExPASy, on the assumption that all cysteines are half-cystines and neglecting any post-translational glycosylation (http://www. expasy.org). The calculated values for wild type TβM containing the histidine tag are as follows: MW ) 69 718 Da, Amg/mL 280nm ) 1.423, 280 ) 99 210 M-1 cm-1. Concentrations determined by Bradford assays were within 5% of values derived from absorbance at 280 nm. Enzyme Activity Assays Using Oxygen Electrode. Steady-state rates of oxygen consumption by WT TβM were measured with a YSI model 5300 biological oxygen electrode, and rate constants were calculated as previously described.15 Assay conditions were identical to those of assay mixtures for HPLC assays. Product Analysis by HPLC. The HPLC methodology was similar to that previously described for product analysis in the reactions of DβM.3 HPLC separations were performed on an Alltech Adsorbosphere reversed phase C-18 column. Octopamine and tyramine were monitored at 224 and 274 nm, respectively. Separation of tyramine and octopamine from other assay components was achieved using a mobile phase of 5 mM acetic acid (pH 5.8), 600 µM heptane sulfonic acid, and 13% methanol at 1 mL/ min; under these conditions octopamine eluted at a retention time of 5.5 min, and tyramine at 15 min. Enzyme Activity Assays. Assay solutions containing 500 µM tyramine, 50 mM ascorbate, 50 mM KPi, 0.1 M KCl, 100-150 µg/mL catalase, and enzyme (0.12-4.4 µM), pH 6, were stirred at 35 °C for periods of 30 min to 13 h, as indicated. CuIISO4 (2-20 µM) was maintained in all assay mixtures at a ratio of g4:1 Cu/ TβM, to ensure that the necessary 2 equiv of Cu were bound to the enzyme. Enzyme was added as the final component of the assay mixtures to initiate the reactions. After a specified amount of time the solutions were quenched with 0.1 M HClO4, spun at 10 000 rpm (5 min) to remove precipitated protein, and filtered (0.2 µm syringe filter, Qiagen) prior to injection onto the HPLC column. For experiments monitoring product formation as a function of time, 100 µL aliquots were removed from the reaction mixtures at various time points during the course of the reaction and added to 1 µL of 70% HClO4. The quenched aliquots were similarly spun and filtered prior to HPLC analysis. Assay samples that were not analyzed by HPLC on the same day as prepared were frozen in liquid nitrogen and stored at -80 °C until subsequent analysis.
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Figure 2. Representative curve illustrating octopamine formation as a function of time in the reaction of WT TβM with tyramine, as determined by HPLC; 50 mM KPi (pH 6), 0.1 M KCl, 50 mM ascorbate, 2 µM CuSO4, 100 µg/mL catalase, 500 µM tyramine, 0.12 µM enzyme; 35 °C.
Figure 1. EPR spectra of WT TβM (34.3 µM) (black), Met471Asp TβM
(37.5 µM) (blue), Met471His TβM (35.4 µM) (brown), Met471Cys TβM (35.8 µM) (green).
Standard Curves. Although both tyramine and octopamine were monitored by HPLC, tyramine displayed much broader peaks and the integrated area for tyramine was highly dependent on the composition of standard solutions. Octopamine peak areas showed almost no deviation for standard curves generated with a sample in which enzyme, Cu, or ascorbate was left out. Product peaks were very reliable, with sharper absorption peaks and greater accuracy. Therefore, reaction progress was monitored by product formation, while the tyramine absorbance was used only qualitatively as a secondary means to confirm substrate turnover by TβM. Solutions for the construction of the product standard curve contained varying amounts of octopamine (50-500 µM) and all other assay components except tyramine, including oxidized WT TβM, to eliminate any background absorbance due to enzyme or other reagents. HClO4 (0.1 M) also was added to the standard curve solutions, and the samples were treated similar to the reaction mixtures, as described above. The fit of the resultant standard curve (y ) 196 256.7x, R2 ) 0.9948) generated from the integrated octopamine peak area (224 nm) was used to quantify the amount of product generated in all reaction mixtures. The validity of the standard curve was verified on each day of experiments, by measuring at least one octopamine-containing sample of known concentration and comparing the integrated area to the standard curves. Control Assays. Control reactions, in which solutions containing all reagents except enzyme and varying amounts of Cu (2-20 µM) were stirred for periods of up to 16 h, also were analyzed by HPLC to establish the background amount of tyramine oxidation in the absence of enzyme. Samples were treated in an identical fashion to enzyme assay mixtures, prior to injection onto the HPLC column. A small peak with the same retention time as that of octopamine occasionally was observed in these control assays suggesting that a minor amount of tyramine was oxidized under these conditions, which was somewhat dependent on the copper concentration. However, the maximum amount of octopamine generated in control assays was 22 µM in solutions containing 20 µM CuII. The final octopamine concentration in the complete reaction of WT TβM (cf. Figure 2) was 10-20% higher than expected in repeated assays, suggesting the presence of an additional reaction product that absorbed in this region, possibly due to ascorbate reaction products. An additional side product could also account
for the inconsistent, low absorbing peaks occasionally observed in control reactions. However, since the identity of any additional products absorbing in this region is unknown, the maximum absorbance in control reactions was used as the background nonenzymatic tyramine oxidation achievable under the assay conditions, representing an upper limit for octopamine generation in the absence of enzyme. TβM Samples for EPR. For the purposes of EPR studies, samples of WT TβM and mutant TβM lacking the Histidine-tag were used, since the histidine tag could bind additional metal ions and thereby preclude accurate determination of the protein EPR spectra and the active site Cu-binding stoichiometries. Details for the removal of the His-tag and for the expression and purification of TβM lacking the His-tag will be reported elsewhere. Preparation of TβM Samples for EPR. TβM samples in 50 mM Tris, 0.1 M NaCl, pH 7.5, were dialyzed against 50 mM Tris, 0.1 M NaCl, 40 µM CuIISO4, pH 7.5, for 4 h. Dialyzed samples (∼2 mg of TβM, V ) 500 µL) were diluted ∼10-fold with copperfree Tris buffer, concentrated down to 1 mL (using Millipore Ultrafree centrifugal concentrators, 10 kDa cutoff membrane), diluted to 5 mL once more with copper-free buffer, and concentrated to a final volume of ∼150 µL, to ensure that the unbound Cu content in the enzyme solutions was
